Research on small RNA gene expression was continued. RNA polymerase (pol) III is responsible for synthesizing tRNAs, 5S rRNA, U6 snRNA and other small transcripts such as Alu RNA. Regulation of pol III reinitiation by the human autoimmuneantigen La was investigated. Alu sequences are small, mobile genetic elements endogenous to the human genome whose RNA-mediated transposition causes insertional mutagenesis in humans. Investigation of Alu retroposon structure and interaction with the Alu RNA binding protein SRP9/14 was continued. This revealed sequences in Alu RNA that severely affect SRP9/14 binding and probably determine retroposon competence. Summary of Major Findings: 1. A model to explain the course of Alu amplification in primates was developed from findings on interactions between Alu RNA and the Alu RNA-binding protein SRP9/14. Alu RNA structure, SRP9/14 binding, and expression in vivo of reconstructed Alu retroposons that were differentially active through primate evolution were analyzed. This revealed that human Alu retroposons have lost a high affinity binding site for SRP9/14 in the right monomer of the dimeric Alu element. This was accompanied by production of Alu left monomer RNA and a decline in the Alu amplification rate that had been noted previously in the human lineage (as compared to primitive primates). This provides the first evidence that points to the Alu sequence itself, as opposed to putative control regions in flanking DNA, as a major determinant of Alu amplification in modern primates and humans. 2. The human autoimmune antigen/phosphoprotein known as La is a transcription termination factor. La controls reinitiation of small RNA genes by also functioning as an (re)initiation cofactor for RNA pol III. The structural regions responsible for La's transcription factor activity were mapped to RRM-2 and a basic region near the C-terminus. RNA concentration-dependent inhibition of this activity in vitro provides a mechanism by which feedback regulation may control small RNA synthesis in vivo. A kinase was purified from HeLa cells that phosphorylates La at a single site adjacent to the C-terminal basic region; this phosphorylation inhibits La's transcription factor activity but not RNA binding. Preliminary evidence is consistent with phosphorylation of this site in vivo.